Prof Mike McPherson

Mike McPherson is Professor of Biochemistry and Molecular Biology in the Institute of Molecular and Cellular Biology and is also a founding member of the Centre for Plant Sciences. He obtained a B.Sc. (I) in Biophysics and Genetics and then a Ph.D. in Genetics from Leeds. After a period of postdoctoral research he was appointed to a Biotechnology lectureship in Leeds in 1985. He held a Leverhulme Trust/Royal Society Senior Research Fellowship in 1993/94.

Research Areas: Protein Engineering; Directed Evolution; Copper Oxidases; Protease Inhibitors; Functional Genomics and Proteomics

Research interests:

The general themes in the laboratory are protein engineering/directed evolution, membrane protein expression for structural proteomics, reverse genetics of plant parasitic nematodes and development of methods for production of nano-structured self-assembling peptides and other bioactive peptides.

? A major research theme concerns copper-containing oxidases, including their catalytic mechanisms, biological roles and the mechanisms of post-translational processing that covert active site amino acids into new organic cofactors. We use mutagenesis, spectroscopic and x-ray crystallography to explore these enzymes.

? We are using a range of high throughput approaches for the cloning and expression of genes encoding membrane proteins from a variety of sources to allow structural analysis.

? Phage and other molecular display technologies are being used in directed evolution programmes including inhibitor and binding proteins. Increasingly we are relying on the development of consensus proteins as a starting point for further molecular diversification and selection of new function in proteins.

? RNAi is being used to identify and validate new target proteins of importance in plant parasitic nematode development.

? We are exploring a range of expression systems for the biological production of peptide materials either for use in self-assembling nano-structured materials for bio nanoscience, industrial or therapeutic applications.

Postgraduate Training Opportunities

Research projects are available on all aspects of the research described below and under each heading project areas are identified.

Research projects:

Copper oxidases are an important class of enzymes that use copper to expand their catalytic potential, allowing difficult chemistries such as C-H bond cleavage. These enzymes often possess modified active site amino acid residues that are formed post-translationally by copper-mediated processing. These modified amino acids represent new organic cofactors. The study of copper oxidases at Leeds benefits from a multi-disciplinary approach involving 3- d structure determination, protein engineering, kinetics and a range of spectroscopy methods to chemistry of model compounds.

Galactose oxidase is a fungal enzyme with an unusual thioether bond linking a cysteine with a tyrosine in the active site. This allows the enzyme to use free radical chemistry to oxidise d -galactose and related alcohols to their corresponding aldehydes with release of hydrogen peroxide. The structure of galactose oxidase was solved in Leeds to 1.7Å resolution and has provided a basis for the molecular analysis of the catalytic mechanism of the enzyme by site-directed mutagenesis and characterisation of variant proteins. We have gained insight into substrate binding, free radical stability and steps in the catalytic mechanism. Intriguingly, galactose oxidase is initially synthesised in a pre-pro-form that undergoes copper-mediated cleavage of the pro region and autocatalytic formation of the cofactor.

Fig 1. Electron density of the C383S variant of galactose oxidase that displays an enhanced K_m for substrate.

1. A project is available to explore the function of pro-region in more detail and identify the novel mechanisms that are associated with copper mediate processing of the enzyme.

2. Projects are available to undertake modification of enzyme properties such as substrate specificity and other properties of the enzyme by a directed evolution approaches. For example galactose oxidase will oxidise fructose poorly but is still used in biosensors for fructose. We have recently identified a mutant form that has enhanced fructose activity and further improvement is required. We have been developing improved E. coli expression systems to facilitate these studies.

Amine oxidases are widely found in nature and the biological functions of the copper-containing amine oxidases include cell adhesion. For example, the human vascular adhesion protein, which is involved in lymphocyte entry in response to inflammation, is actually an amine oxidase.

We have focused on the enzyme from Escherichia coli as a good model system. The copper amine oxidases contain an active site tyrosine that is modified by a copper and oxygen dependent process to trihydroxyphenylalanine quinone (TPQ) that acts as redox active cofactor. The crystal structure of the E. coli enzyme was solved in Leeds in 1995 and has guided site-directed mutagenesis to explore the roles of several residues in the catalytic mechanism. An important technique is cryocrystallographic trapping of reaction intermediates. This approach has been used to studying oxygen binding during the oxidative half reaction.

Fig 2. Structure of E. coli amine oxidase active site complexed with the suicide inhibitor 2-hydrazinopyridine (A) in the wild-type enzyme (B) in the Y369F variant showing the large rotational change in the adduct position which is now associated with the copper centre.

We have also undertaken studies of plant amine oxidases and have cloned amine oxidase genes of Arabidopsis thaliana to define the pat terns of gene expression and to identify the biological roles of these molecules.

3. Projects are available to explore the role of residues that impact on oxygen binding and chemistry in amine oxidases.

4. A project is available to explore inter-subunit communication between the monomers of the dimer of amine oxidase through the production of heterodimers and mutagenesis studies.

Lysyl oxidases

Lysyl oxidases are involved in extracellular matrix cross-lining and associated diseases. In mammals the lysyl oxidase gene is an anti-oncogene. These enzymes represent a special class of the copper amine oxidases. They also bind copper, but are monomeric and undergo post-translational modification to form a lysyltyrosyl quinone cofactor involving the oxidation of an active site tyrosine and the cross-linking with an adjacent lysine side chain. Since these enzymes are found in the extracellular matrix environment they are less soluble that most proteins and have proven extremely difficult to express in standard expression systems. We are currently focusing on this class of enzyme by using a synthetic gene approach for expression studies. We have had success in generating soluble protein and will be undertaking structural studies.

5. Projects are available for further expression, protein purification, characterisation and structural studies leading to a programme of mutagenesis and protein engineering.

Membrane protein structural proteomics

Membrane proteins represent a major class of proteins encoded within genomes and in humans represent around 50% of current drug targets. Structural studies of membrane proteins are extremely underdeveloped primarily due to the unusual nature of the proteins and associated difficulties in high level expression, purification and crystallization. We are developing systems for high throughput expression and purification of membrane proteins based on a variety of expression systems using robotic approaches. The critical issue is correct functional expression. We are examining a wide variety of proteins from various sources including prokaryotic and eukaryotic. Part of this work is funded under the Membrane Protein Structure Initiative funded by BBSRC with a major grant to Leeds and seven other partners in the UK .

6. Projects are available involving cloning, expression, characterisation and crystallization of membrane proteins using a range of appropriate systems.

Consensus proteins and display approaches

We are using consensus protein design from multiple sequence alignments to generate new proteins that provide a structural framework for developing new biologically relevant proteins. In some cases consensus proteins function effectively without further optimisation. In other cases, particularly with binding proteins there is a need to introduce further diversity to generate libraries from which good binders can be selected. We are using different display technologies to develop such libraries for selection of new proteins. For example some are being developed to link in with the need to inhibit plant nematode proteins (see below)

7. Projects are available to develop and characterise new consensus proteins and to generate display libraries for functional selection of new functionalities.

Plant/nematode interaction

Plant nematodes represent a major agronomic problem leading to dramatic crop loss and requiring highly toxic nematicide control regimes. We are interested in understanding molecular events that occur during the parasitic interactions and in developing environmentally acceptable approaches for the control of nematode pests. One successful approach developed in Leeds is the interference with nematode cysteine proteases using cystatins, small protein inhibitors of these proteases. We are currently exploring the use of directed evolution to improve inhibitor function. We are using phage display and other display approaches to identify proteins and peptides that inhibit the action of new nematode derived targets and that may provide new lead compounds as environmentally friendly nematicides. For example we have isolated peptides that mimic the effects of anthelmintic drugs and nematicides and there are many more potential targets that might be explored.

A key technique that allows the validation of target genes is RNAi in which double stranded RNA molecules representing part of the target gene are introduced into the nematodes. A methods developed in Leeds allows the uptake of RNAi by J2 stage plant nematodes that do not feed, but can be induced to ingest material including dsRNA. We have been using this approach to explore the role of specific genes in extracellular matrix development and have identified genes that affect normal nematode development.

Fig. 3 Effect of RNAi on development of the extracellular matrix of C. elegans showing extremely abnormal development.

8. Projects are available on the development of RNAi tools for molecular analysis and plant expression studies.

Bioproduction of peptides and bionanoscience

In association with the Centre for Self Organising Molecular Systems at Leeds we are developing approaches for generic and large scale production peptides and peptide arrays that have self-assembling properties that respond to environmental triggers. These materials can be used to form products including responsive gels, foams and fibres. We are also interested in the expansion of these approaches for the production of therapeutic or agrochemically relevant peptidic materials. Currently we are using microbial production systems and are beginning to focus on plant-based production. Such systems will also be of importance in the production of other types to peptide based pharmaceuticals.